Tumor necrosis factor (TNF) alpha is a cytokine that promotes the inflammation and its associated signs by binding to its receptor. It is produced by macrophages and many other immune cells. It is involved in pathogenesis of many inflammatory disorders like rheumatoid arthritis, psoritic arthritis, SLE, Crohn's disease etc. Hohmann et al (Hohmann et al. 1989 J Biol Chem. 25, 14927-34) identified 2 distinct receptors of TNF-alpha which are present on different cell types viz. myeloid cells and epithelial cells. Using monoclonal antibodies, Brockhaus et al (Brockhaus et al. 1990 Proc Natl Acad Sci U S A., 87(8), 3127-31) demonstrated that both TNF-alpha and beta bind to both the receptors with high affinity.
Tumor necrosis factor-alpha (TNF-alpha) is a central regulator of inflammation, and TNF-alpha antagonists may be effective in treating inflammatory disorders in which TNF-alpha plays an important pathogenetic role. Inhibition of TNF has proven to be an effective therapy for patients with rheumatoid arthritis and other forms of inflammatory disease including psoriasis, psoriatic arthritis, and ankylosing spondylitis, inflammatory bowel disease. One such TNF-alpha antagonist is Etanercept.
Etanercept is a dimeric fusion protein produced by recombinant DNA technology where gene of soluble, ligand binding portion of TNF receptor 2 is fused with gene of Fc component of human IgG1 to give the desired fusion protein (U.S. Pat. No. 7,648,702). Etanercept is expressed in CHO cells. The Fc component of Etanercept lacks CH1 domain but has CH2, CH3 domains and hinge region. The fusion protein has approximate molecular weight of 150 kD and consists of 934 amino acids. Etanercept interferes with TNF and acts as a TNF inhibitor due to which it can be used as a biopharmaceutical to treat autoimmune diseases. It prevents progressive destruction of joints in patients with rheumatoid arthritis and the arthritis of psoriasis.
Due to its unique structure, Etanercept binds 50-100 folds more efficiently to TNF alpha than its endogenous receptor (Gofeeet al. 2003 J Am Acad Dermatol. 49, S105-111, Strober 2005 Semin Cutan Med Surg. 24; 28-36). Additionally, due to its dimeric nature it can bind to 2 TNF alpha molecules as compared to one bound by endogenous receptor. Conjugation of this molecule to Fc region of IgG increases the half life as compared to endogenous soluble form. Commercially, Etanercept is available in both Lyophilized and liquid forms.
The most important feature of a composition is to help the protein to retain its structural conformation or its activity. The stability of protein in a composition can be related with long-term storage. It is understood to mean that the active polypeptide of the pharmaceutical composition does not substantially lose its activity as compared to the composition at the beginning of storage.
All polypeptides have an Isoelectric Point (pI), which is generally defined as the pH at which a polypeptide carries no net charge. It is known in the art that protein solubility is typically lowest when the pH of the solution is equal to the isoelectric point (pI) of the protein.
The Tm of the Fab domain of a protein is a good indicator of the thermal stability of a protein and may further provide an indication of the shelf-life. Tm values of proteins determined by differential scanning calorimetry, give insight into heat-induced changes in protein conformation, mechanisms of protein unfolding and stabilization in solution. A lower Tm indicates less stability of a protein in given solution, whereas a higher Tm indicates a better stability of the protein. The Tm of the protein will vary based on the formulation composition which in turn reflects its stability in respective formulation.
During long term storage, both aqueous and lyophilized compositions of proteins can lose active protein due to aggregation or degradation. Aggregation of the protein can lead to immunogenicity and is undesirable. Since the concentration of Etanercept used in the composition is high, there is a likely possibility of protein aggregation during long term storage. To improve the stability of the protein either the concentration of the existing excipients can be varied or new excipients can be added to modify the composition.
U.S. Pat. Nos. 5,215,743; 7,648,702; US application US 20070053906 and WO 2011141926 disclose pharmaceutical compositions comprising aqueous composition of TNF-binding protein comprising a TNF-binding protein, a buffer and an isotonicity agent.